Energy balance (involving energy metabolism, nutritional state, lipid storage and the like) is an important criteria for health. This energy homeostasis involves food intake and metabolism of carbohydrates and lipids to generate energy necessary for voluntary and involuntary functions. Metabolism of proteins can lead to energy generation, but preferably leads to muscle formation or repair. Among other consequences, a lack of energy homeostasis lead to over or under formation of adipose tissue.
Formation and storage of fat is insulin-modulated. For example, insulin stimulates the transport of glucose into cells, where it is metabolized into xcex1-glycerophosphate which is used in the esterification of fatty acids to permit storage thereof as triglycerides. In addition, adipocytes (fat cells) express a specific transport protein that enhances the transfer of free fatty acids into adipocytes.
Adipocytes also secrete several proteins believed to modulate homeostatic control of glucose and lipid metabolism. These additional adipocyte-secreted proteins include adipsin, complement factors C3 and B, tumor necrosis factor xcex1, the ob gene product and Acrp30. Evidence also exists suggesting the existence of an insulin-regulated secretory pathway in adipocytes. Scherer et al., J. Biol. Chem. 270(45): 26746-9, 1995. Over or under secretion of these moieties, impacted in part by over or under formation of adipose tissue, can lead to pathological conditions associated directly or indirectly with obesity or anorexia.
Acrp30 is a 247 amino acid polypeptide that is expressed exclusively by adipocytes. The Acrp30 polypeptide is composed of a amino-terminal signal sequence, a 27 amino acid stretch of no known homology, 22 perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa collagen repeats and a carboxy terminal globular domain. See, Scherer et al. as described above and International Patent Application No. WO 96/39429. Acrp30, an abundant human serum protein regulated by insulin, shares structural similarity, particularly in the carboxy-terminal globular domain, to complement factor Clq and to a summer serum protein of hibernating Siberian chipmunks (Hib27). Expression of Acrp30 is induced over 100-fold during adipocyte differentiation. Acrp30 is suggested for use in modulating energy balance and in identifying adipocytes in test samples.
Another secreted protein that appears to be exclusively produced in adipocytes is apM1, described, for example, in Maeda et al., Biochem. Biophys. Res. Comm. 221: 286-9, 1996. A 4517 bp clone had a 244 amino acid open reading frame and a long 3xe2x80x2 untranslated region. The protein included a signal sequence, an amino-terminal non-collagenous sequence, 22 collagen repeats (Gly-XAA-Pro or Gly-Xaa-Xaa), and a carboxy-terminal region with homology to collagen X, collagen VIII and complement protein Clq.
Complement factor Clq consists of six copies of three related polypeptides (A, B and C chains), with each polypeptide being about 225 amino acids long with a near amino-terminal collagen domain and a carboxy-terminal globular region. Six triple helical regions are formed by the collagen domains of the six A, six B and six C chains, forming a central region and six stalks. A globular head portion is formed by association of the globular carboxy terminal domain of an A, a B and a C chain. Clq is therefore composed of six globular heads linked via six collagen-like stalks to a central fibril region. Sellar et al., Biochem. J. 274: 481-90, 1991. This configuration is often referred to as a bouquet of flowers. Acrp30 has a similar bouquet structure formed from a single type of polypeptide chain.
Clq has been found to stimulate defense mechanisms as well as trigger the generation of toxic oxygen species that can cause tissue damage (Tenner, Behring Inst. Mitt. 93:241-53, 1993). Clq binding sites are found on platelets. Additionally complement and Clq play a role in inflammation. The complement activation is initiated by binding of Clq to immunoglobulins.
Inhibitors of Clq and the complement pathway would be useful for anti-inflammatory applications, inhibition of complement activation and thrombotic activity.
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 40-285 of SEQ ID NO:2, wherein the sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxyl-terminal Clq domain comprises 10 beta strands. Within one embodiment the polypeptide that is at least 90% identical in amino acid sequence to residues 16-285 of SEQ ID NO:2. Within another embodiment the collagen domain consists of 24 Gly-Xaa-Xaa repeats and 10 Gly-Xaa-Pro repeats. Within another embodiment the carboxyl-terminal Clq domain comprises the sequence of SEQ ID NO:5. Within another embodiment the carboxy-terminal Clq domain comprises amino acid residues 151-155, 172-174, 180-183, 187-190, 193-205, 208-214, 220-227, 229-241, 246-251 and 269-274 of SEQ ID NO:2. Within another embodiment any differences between said polypeptide and SEQ ID NO:2 are due to conservative amino acid substitutions. Within another embodiment the polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2. Within a further embodiment the polypeptide comprises residues 16-285 of SEQ ID NO:2. Within another embodiment the polypeptide is covalently linked at the amino or carboxyl terminus to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores. Within yet another embodiment the collagen domain consists of amino acid residues 41-141 of SEQ ID NO:2. Within another embodiment the carboxy-terminal Clq domain consists of amino acid residues 142-285 of SEQ ID NO:2.
The invention also provides an isolated polypeptide selected from the group consisting of: a) a polypeptide consisting of a sequence of amino acid residues that is 75% identical in amino acid sequence to amino acid residue 40 to amino acid residue 141 of SEQ ID NO:2; b) a polypeptide consisting of a sequence of amino acid residues that is 75% identical in amino acid sequence to amino acid residue 142 to amino acid residue 285 of SEQ ID NO:2; and c) a polypeptide consisting of a sequence of amino acid residues that is 75% identical in amino acid sequence to amino acid residue 40 to 285 of SEQ ID NO:2.
Within another aspect, the invention provides a fusion protein comprising a first portion and a second portion joined by a peptide bond, the first portion consisting of a polypeptide selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residue 16 to amino acid residue 285 of SEQ ID NO:2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue 281; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 16 to amino acid residue 285; d) a portion of the zacrp2 polypeptide as shown in SEQ ID NO:2, comprising the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; e) a portion of the zacrp2 polypeptide as shown in SEQ ID NO:2, comprising the Clq domain or an active portion of the Clq domain; or f) a portion of the zacrp2 polypeptide as shown in SEQ ID NO:2 comprising of the collagen-like domain and the Clq domain; and the second portion comprising another polypeptide. Within one embodiment the first portion is selected from the group consisting of: a) a polypeptide consisting of the sequence of amino acid residue 40 to amino acid residue 141 of SEQ ID NO:2; b) a polypeptide consisting of the sequence of amino acid residue 142 to amino acid residue 285 of SEQ ID NO:2; c) a polypeptide consisting of the sequence of amino acid residue 40 to 285 of SEQ ID NO:2.
Within another aspect, the invention provides a polypeptide as described above; in combination with a pharmaceutically acceptable vehicle.
Within another aspect is provided an antibody or antibody fragment that specifically binds to a polypeptide as described above. Within one embodiment the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b); and d) human monoclonal antibody. Within another embodiment the antibody fragment is selected from the group consisting of F(abxe2x80x2), F(ab), Fabxe2x80x2, Fab, Fv, scFv, and minimal recognition unit. Within another embodiment is provided an anti-idiotype antibody that specifically binds to the antibody described above.
Within another aspect, the invention provides an isolated polynucleotide selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 40-285 of SEQ ID NO:2, wherein the sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxyl-terminal Clq domain comprising 10 beta strands. Within one embodiment the polypeptide is at least 90% identical in amino acid sequence to residues 16-285 of SEQ ID NO:2. Within another embodiment the collagen domain consists of 24 Gly-Xaa-Xaa repeats and 10 Gly-Xaa-Pro repeats. Within yet another embodiment the carboxyl-terminal Clq domain comprises the sequence of SEQ ID NO:5. Within another embodiment the carboxy-terminal Clq domain comprises amino acid residues 151-155, 172-174, 180-183, 187-190, 193-205, 208-214, 220-227, 229-241, 246-251 and 269-274 of SEQ ID NO:2. Within another embodiment any differences between said polypeptide and SEQ ID NO:2 are due to conservative amino acid substitutions. Within another embodiment the polynucleotide encodes a polypeptide that specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2. Within still another embodiment the polynucleotide encodes a polypeptide that comprises residues 16-285 of SEQ ID NO:2. Within another embodiment the collagen domain consists of amino acid residues 41-141 of SEQ ID NO:2. Within yet another embodiment the carboxy-terminal Clq domain consists of amino acid residues 142-285 of SEQ ID NO:2.
Also provided is an isolated polynucleotide selected from the group consisting of: a) a sequence of nucleotides from nucleotide 1 to nucleotide 1161 of SEQ ID NO:1; b) a sequence of nucleotides from nucleotide 133 to nucleotide 987 of SEQ ID NO:1; c) a sequence of nucleotides from nucleotide 178 to nucleotide 987 of SEQ ID NO:1; d) a sequence of nucleotides from nucleotide 250 to nucleotide 987 of SEQ ID NO:1; e) a sequence of nucleotides from nucleotide 556 to nucleotide 987 of SEQ ID NO:1; f) a sequence of nucleotides from nucleotide 133 to nucleotide 555 of SEQ ID NO:1; g) a sequence of nucleotides from nucleotide 178 to nucleotide 555 of SEQ ID NO:1; h) a sequence of nucleotides from nucleotide 250 to nucleotide 555 of SEQ ID NO:1; i) a polynucleotide encoding a polypeptide, the polypeptide consisting of a sequence of amino acid residues that is at least 75% identical to a polypeptide consisting of the amino acid sequence of residues 40 to 141 of SEQ ID NO:2; j) a polynucleotide encoding a polypeptide, the polypeptide consisting of a sequence of amino acid residues that is at least 75% identical to a polypeptide consisting of the amino acid sequence of residues 142 to 285 of SEQ ID NO:2; k) a polynucleotide encoding a polypeptide, the polypeptide consisting of a sequence of amino acid residues that is at least 75% identical to a polypeptide consisting of the amino acid sequence of residues 40 to 285 of SEQ ID NO:2; l) a polynucleotide encoding a polypeptide consisting of a sequence of amino acid residues that is at least 75% identical to a polypeptide consisting of the amino acid sequence of residues 16 to 141 of SEQ ID NO:2; m) a polynucleotide that remains hybridized following stringent wash conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1, or the complement of SEQ ID NO:1; n) nucleotide sequences complementary to a), b), c), d), e), f), g), h), i), j), k), l) or m) and o) degenerate nucleotide sequences of i), j), k) or l).
The invention further provides an isolated polynucleotide encoding a fusion protein comprises a first portion and a second portion joined by a peptide bond, the first portion is selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residues 40 to 285 of SEQ ID NO:2; b) a polypeptide comprising the sequence of amino acid residues 1 to 285 of SEQ ID NO:2; c) a polypeptide comprising the sequence of amino acid residues 16 to 285 of SEQ ID NO:2; d) a portion of a polypeptide of SEQ ID NO:2 comprising the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; e) a portion of the polypeptide of SEQ ID NO:2 containing the Clq domain; or f) a portion of the polypeptide of SEQ ID NO:2 including the collagen-like domain and the Clq domain; and the second portion comprising another polypeptide.
Also provided is an isolated polynucleotide consisting of the sequence of nucleotide 1 to nucleotide 855 of SEQ ID NO:10.
Within another aspect, the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator. Within one embodiment the DNA segment encodes a polypeptide that is at least 90% identical in amino acid sequence to residues 16-285 of SEQ ID NO:2. Within another embodiment the collagen domain consists of 24 Gly-Xaa-Xaa repeats and 10 Gly-Xaa-Pro repeats. Within another embodiment the carboxyl-terminal Clq domain comprises the sequence of SEQ ID NO:5. Within another embodiment the carboxy-terminal Clq domain comprises amino acid residues 151-155, 172-174, 180-183, 187-190, 193-205, 208-214, 220-227, 229-241, 246-251 and 269-274 of SEQ ID NO:2. Within another embodiment any differences between said polypeptide and SEQ ID NO:2 are due to conservative amino acid substitutions. Within another embodiment the collagen domain consists of amino acid residues 41-141 of SEQ ID NO:2. Within another embodiment the carboxy-terminal Clq domain consists of amino acid residues 142-285 of SEQ ID NO:2. Within yet another embodiment the polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2. Within yet another embodiment the DNA segment encodes a polypeptide comprising residues 16-285 of SEQ ID NO:2. Within a further embodiment the DNA segment encodes a polypeptide covalently linked at the amino or carboxyl terminus to an affinity tag. Within another embodiment the DNA segment further encodes a secretory signal sequence operably linked to the polypeptide. Within a related embodiment the secretory signal sequence comprises residues 1-15 of SEQ ID NO:2.
Within another aspect, the invention provides a cultured cell into which has been introduced an expression vector as described above, wherein the cell expresses the polypeptide encoded by the DNA segment.
Within still another aspect, the invention a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector as described above; whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.